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Research ArticleCardiovascular
Open Access

Histone Deacetylase Inhibitors Relax Mouse Aorta Partly through Their Inhibitory Action on L-Type Ca2+ Channels

Changbo Zheng, Mingkui Zhong, Zenghua Qi, Fan Shen, Qiannan Zhao, Lulu Wu, Yu Huang, Suk-Ying Tsang and Xiaoqiang Yao
Journal of Pharmacology and Experimental Therapeutics November 2017, 363 (2) 211-220; DOI: https://doi.org/10.1124/jpet.117.242685

Abstract

Histone deacetylase (HDAC) inhibitors modulate acetylation/deacetylation of histone and nonhistone proteins. They have been widely used for cancer treatment. However, there have been only a few studies investigating the effect of HDAC inhibitors on vascular tone regulation, most of which employed chronic treatment with HDAC inhibitors. In the present study, we found that two hydroxamate-based pan-HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), could partially but acutely relax high extracellular K+-contracted mouse aortas. SAHA and TSA also attenuated the high extracellular K+-induced cytosolic Ca2+ rise and inhibited L-type Ca2+ channel current in whole-cell patch-clamp. These data demonstrate that SAHA could inhibit L-type Ca2+ channels to cause vascular relaxation. In addition, SAHA and TSA dose dependently relaxed the arteries precontracted with phenylephrine. The relaxant effect of SAHA and TSA was greater in phenylephrine-precontracted arteries than in high K+-contracted arteries. Although part of the relaxant effect of SAHA and TSA on phenylephrine-precontracted arteries was related to L-type Ca2+ channels, both agents could also induce relaxation via a mechanism independent of L-type Ca2+ channels. Taken together, HDAC inhibitors SAHA and TSA can acutely relax blood vessels via their inhibitory action on L-type Ca2+ channels and via another L-type Ca2+ channel-independent mechanism.

Introduction

Histone deacetylases (HDACs) are a family of enzymes that regulate protein activity through removal of acetyl groups (Richardson et al., 2017). A total of 18 histone deacetylases can be grouped into four classes (I, II, III, and IV). The most well known molecular targets of HADCs are the histone proteins that compact DNA in chromatin. Reversible acetylation and deacetylation of histone proteins in chromatin constitutes a major epigenetic mechanism for the control of gene expression. In addition, up to 1750 nonhistone target proteins of HADCs have been identified (Choudhary et al., 2009). It has been suggested that acetylation and deacetylation of these nonhistone proteins may regulate such crucial cellular processes as the cell cycle, cytoskeleton reorganization, endocytosis, and vesicular trafficking (Choudhary et al., 2009). Owing to the crucial importance of HADC activity in physiologic and pathologic processes, HADC inhibitors have been developed as therapeutic options for a variety of diseases, including cancer, mood disorders, and HIV (Dokmanovic et al., 2007). In an experimental model, HDAC inhibitors have also been shown to be beneficial in arrhythmia, myocardial infraction, hypertension, and atherosclerosis (Eom and Kook, 2014). HDAC inhibitors can be divided into several structural classes, including hydroxamates, cyclic peptides, and others (Dokmanovic et al., 2007). Suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) are hydroxamate-based pan-HDAC inhibitors that inhibit class I and class II HDAC proteins (Dokmanovic et al., 2007). However, their inhibitory potency to different classes of HDAC proteins varies greatly (Ko et al., 2013). Of the two, only SAHA has been approved by the FDA for clinical use, for treatment of cutaneous T-cell lymphoma and multiple myeloma (Dokmanovic et al., 2007).

Only a few studies have investigated the action of HDAC inhibitors in vascular tone control. Most examined the effect of chronic treatment with HDAC inhibitors on vascular tone regulation, but the results are controversial. Usui et al. (2012) showed that chronic treatment of rat mesenteric arteries with TSA for 3 weeks enhanced the endothelium-dependent relaxation and reduced the angiotensin II (Ang II)-induced contraction in spontaneously hypertensive rats. In contrast, several other studies reported that chronic treatment with TSA reduced the endothelium-dependent and endothelium-independent vascular relaxation (Colussi et al., 2012; Mathews et al., 2014; Pandey et al., 2014). These authors found that, whereas the endothelium-dependent component could be attributed to TSA actions on the expressional level of arginase 2 and nitric oxide synthase (Mathews et al., 2014), the endothelium-independent components might be related to acetylation/deacetylation of such smooth muscle contractile proteins as myosin light chain and smooth muscle actin (Colussi et al., 2012). Interestingly, a recent study also demonstrated an acute effect of HDAC inhibitors on vascular tone. In that study, acute treatment of rat aortic rings with 10 μM TSA for 15 minutes substantially reduced Ang II-induced contraction, probably via an inhibitory action of TSA on p66shc phosphorylation in vascular smooth muscle cells (Kang et al., 2015).

In the present study, we aimed to investigate acute effects of HDAC inhibitors on vascular tone. The results show that application of two commonly used HDAC inhibitors, SAHA and TSA, could induce an acute relaxation in high extracellular K+- and phenylephrine-precontracted rat aortic rings. This relaxation was endothelium-independent. Ca2+ influx measurement and whole-cell patch-clamp studies demonstrated that SAHA and TSA inhibit the activity of L-type Ca2+ channels in rat vascular smooth muscle cells.

Material and Methods

Chemicals.

Verapamil, methylene blue, and phenylephrine were from Sigma-Aldrich (St. Louis, MO). SAHA (PubChem CID:5311), TSA (PubChem CID:444732), LBH-589 (PubChem CID:Panobinostat) (6918837), FK228 (romidepsin) (PubChem CID:5352062), and PDX-101 (belinostat) (PubChem CID:6918638) were from Selleckchem (Houston, TX). U46619, SAHA, TSA, LBH-589, FK228, and PDX-101 were dissolved in dimethyl sulfoxide (DMSO), and other drugs were prepared in distilled water.

Measurement of Vascular Tension.

All animals were obtained from the Laboratory of Animal Services Center, the Chinese University of Hong Kong, Hong Kong, China. The animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health. Isometric tension of aortic rings were recorded by Multi Wire Myograph System (Danish Myo Technology A/S, Aarhus, Denmark) as described (Tian et al., 2012). Briefly, male C57 adult mice were killed by carbon dioxide suffocation. After euthanasia, the mouse thoracic aortas were quickly dissected free and cut into 2-mm long rings in ice-cold and oxygenated Krebs buffer, which contained in mM: 119 NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 d-glucose. The vascular rings were mounted onto two thin stainless-steel holders in a myograph filled with Krebs solution bubbled with 95% O2 and 5% CO2 at 37°C. The optimum baseline tension was set at 3 mN. After an equilibration period and 60 mM KCl solution pretreatment (which contained in mM: 63.7 NaCl, 60 KCl, 2.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1.2 KH2PO4, and 11 d-glucose), the rings were exposed to 1 μM phenylephrine to test their contractile responses and subsequently challenged with acetylcholine to certify the integrity of endothelium.

Preparation of Acutely Isolated Smooth Muscle Cells.

The smooth muscle cells were freshly dispersed as described previously (Eto et al., 2001). Briefly, male C57 adult mice were anesthetized with ether. The aorta was dissected. The connective tissue and the endothelium were carefully removed under microscope and the smooth muscle was cut into 1- to 2-mm sections. The tissues were preincubated with oxygenated Ca2+-free phosphate-buffered saline (PBS) with collagenase II (2% w/v in 1× PBS) and trypsin (0.25% w/v) for 40 minutes at 18–22°C, and the smooth muscle was cut into one to two arterial branches and dissected. The tissues were washed three times with oxygenated Ca2+-free PBS and then gently agitated with a blunt-tipped glass pipette until the solution became cloudy. The debris was then removed with a fine 200-μm nylon mesh. The cell suspension was stored at 4°C and used within 5 hours of cell harvesting.

Preparation of Smooth Muscle Cell Line.

An aortic smooth muscle cell line from rat (A7r5) was obtained from the American Type Culture Collection (ATCC). The A7r5 cell line was cultured in 90% Dulbecco’s modified Eagle’s medium (Gibco/Thermo Fisher Scientific, Gaithersburg, MD), and 10% fetal bovine serum (Gibco/Thermo Fisher Scientific) at 37°C and 5% CO2 in air.

Cytosolic Ca2+ Measurement.

The cytosolic Ca2+ was measured as described elsewhere (Qi et al., 2015). Briefly, smooth muscle cells were loaded with Fluo-4/AM (Invitrogen/Thermo Fisher Scientific, Waltham, MA) in normal Tyrode’s solution that contained in mM: 140 NaCl, 5.4 KCl, 1 MgCl2, 2 CaCl2, 5.5 glucose, and 5 HEPES, pH 7.4. To deplete intracellular Ca2+ stores, the cells were treated with 4 μM thapsigargin in Ca2+-free Tyrode’s solution, which contained in mM: 140 NaCl, 5.4 KCl, 1 MgCl2, 5.5 glucose, 0.2 ethylene glycol tetraacetic acid (EGTA), and 5 HEPES, pH 7.4. Store-operated Ca2+ entry (SOCE) was initiated by the addition of 2 mM Ca2+ to the bath. The real-time fluorescent images were captured every 10 seconds and analyzed by MetaFluor imaging software (Molecular Devices, Sunnyvale, CA).

Patch-Clamp Recording.

For whole-cell patch-clamp recordings, single A7r5 smooth muscle cells were seeded on glass coverslips precoated for 30 minutes with Matrigel containing 50 μg/ml laminin (Corning, Corning, NY) in 0.1% gelatin (Sigma-Aldrich). The glass coverslips containing the cells were placed into a room-temperature–recording chamber and perfused continuously with Tyrode’s solution, which contained in mM: 140 NaCl, 1 MgCl2, 1.8 CaCl2, 5.4 KCl, 10 glucose, and 5 HEPES, pH adjusted to 7.4 by NaOH. The whole-cell patch-clamp recordings (voltage- and current-clamp configurations) were performed with an Axopatch 200B amplifier (Molecular Devices) and pCLAMP 10.4 software (Molecular Devices). Signals were digitized at 10 kHz and filtered at 2 kHz. Microelectrodes (1B150F-4; World Precision Instruments, Sarasota, FL) with resistances of 3–7 MΩ were pulled from P-97 puller (Sutter Instrument, Novato, CA). L-type Ca2+ current was recorded from single cells with ruptured whole-cell patch-clamp techniques. After establishing patch-clamp, the Tyrode’s solution was replaced with the Ba2+-containing solution, which contained in mM: 108 NaCl, 20 triethylamine (TEA)-Cl, 5.4 CsCl, 10 BaCl2, 1 MgCl2, 5.5 glucose, and 10 HEPES, pH adjusted to 7.4 by NaOH. The pipette solution contained in mM: 115 Cs-aspartate, 20 TEA-Cl, 1 MgCl2, 5 BAPTA, 3 Mg·ATP, 0.2 GTP, and 10 HEPES, pH 7.4 adjusted with CsOH (Ochi et al., 2016). At a holding potential of −80 mV, current-voltage relations were obtained by applying 500-millisecond depolarization steps in 10-mV increments at 0.2 Hz from −40 to 50 mV. The test potential initially depolarized from –80 to –40 mV to detect and inactivate the T-type Ca2+ channel current.

Statistical Analysis.

Results are expressed as means ± S.E.M. The relaxation was presented as percentage of the evoked contraction. Data were analyzed using GraphPad Prism software. Two-tailed Student’s t test was used for two group comparison. One-way analysis of variance with Newman-Keuls post-hoc test were used for statistical evaluation when more than two groups were compared. P < 0.05 was taken as statistically significant.

Results

HDAC Inhibitors Reversed the Vascular Contraction Elicited by High Extracellular K+ in Mouse Aortas.

Exposure of aortic segments to high extracellular K+ concentration at 60 mM induced a rapid contraction of mouse aortas, which was completely reversed by an L-type Ca2+ channel inhibitor verapamil at 10 μM (Fig. 1A). SAHA at 3–300 μM or TSA at 1–100 μM induced dose-dependent relaxation in the arteries that were precontracted with high extracellular K+ (Fig. 1, B and C). Three hundred micromolar SAHA and 100 μM TSA resulted in a relaxation of 45% ± 2% and 47% ± 3%, respectively (Fig. 1, B and C). The effect of SAHA and TSA was endothelium-independent (Fig. 1, B and C).

Fig. 1.
Fig. 1.

SAHA and TSA reversed the vascular contraction elicited by high extracellular K+ in mouse aortas. (A) Representative traces (left) and summarized data (right) showing verapamil-induced relaxation in the arteries precontracted by 60 mM KCl. The vehicle control (0.1% DMSO) had no effect. (B and C) The vascular relaxant effect of SAHA (B) and TSA (C) on 60 mM KCl-precontracted aortas with endothelium (+Endo) or without endothelium (−Endo). Representative traces (left) and data summary (right) of relaxing effect are shown. The data are expressed as mean ± S.E.M. (n = 5). ***P < 0.001 vs. vehicle (DMSO) control.

Several other HDAC inhibitors were also tested, including hydroxamates LBH-589 and PDX-101, as well as cyclic peptide FK-228. Like SAHA and TSA, all these HDAC inhibitors caused dose-dependent relaxation in high extracellular K+-contracted arteries (Supplemental Fig. 1).

TSA Attenuated the High Extracellular K+-Elicited Cytosolic Ca2+ Rise and Inhibited L-Type Ca2+ Channel Current.

Cytosolic Ca2+ rise in response to high extracellular K+ was measured in the freshly isolated mouse vascular smooth muscle cells. Sixty millimolar extracellular K+ stimulated Ca2+ influx in these cells. Three hundred micromolar SAHA or 100 μM TSA markedly attenuated the Ca2+ influx elicited by high extracellular K+ (Fig. 2A).

Fig. 2.
Fig. 2.

SAHA and TSA attenuated the high extracellular K+-elicited cytosolic Ca2+ rise and inhibited L-type Ca2+ channel current. (A) Effect of SAHA and TSA on high K+-induced Ca2+ rise. Acutely isolated smooth muscle cells were treated with SAHA/TSA in normal Krebs solution bath, followed by a change to 60 mM KCl Krebs solution bath, which induced a Ca2+ rise. SAHA/TSA at 300/100 μM was applied about 5 minutes before high K+ treatment. Representative traces (left) and data summary (right) of Ca2+ responses are shown. (B) Effect of SAHA and TSA on ionomycin-induced Ca2+ rise. Acutely isolated smooth muscle cells were treated with TSA/SAHA in normal Krebs solution bath, followed by Krebs solution bath with 1 μM ionomycin, which induced a Ca2+ rise. SAHA/TSA at 300/100 μM was applied about 5 minutes before ionomycin treatment. Representative traces (left) and data summary (right) of Ca2+ responses are shown. (C and D) Whole-cell current recording in A7r5 cells. Shown are current-voltage (I–V) relationships (left), voltage protocol and representative whole-cell current traces (right) of A7r5 cells with or without SAHA (C) and TSA (D). SAHA/TSA at 300/100 μM was applied about 5 minutes before current recording. The data are expressed as the mean ± S.E.M. [n = 5 in (A and B), n = 6 in (C and D)]. *P < 0.05; ***P < 0.001 vs. vehicle (DMSO) control.

Literature shows that hydroxamates such as TSA and SAHA are able to chelate multiple ions, including Zn2+, Ni2+, and Fe3+ (Hebbel et al., 2010; Griffith et al., 2011). In our experiments, 300 μM SAHA or 100 μM TSA had no significant effect on 1 μM ionomycin-induced cytosolic Ca2+ rise in vascular smooth muscle cells (Fig. 2B), suggesting that the ability of these agents in chelating/buffering cytosolic Ca2+ was minimal, if any, in our experimental conditions.

Under high extracellular K+, L-type Ca2+ channel current is the major source of Ca2+, which initiates contraction of arterial smooth muscle cells and regulates blood pressure (Zhang et al., 2007). Whole-cell patch-clamp was used to measure L-type Ca2+channel current. Because of technical difficulty in making giga-ohm seal on the plasma membrane of freshly isolated mouse smooth muscle cells, we opted to use A7r5 rat smooth muscle cells for patch-clamp studies. In whole-cell recording, the cells were held at −80 mV and voltage steps ranging from −40 to +50 mV were applied for 500 milliseconds in 10 mV step increments. A typical U-shaped voltage-gated Ca2+ current was recorded in these cells (Fig. 2, C and D) (Sun et al., 2000). Application of 300 μM SAHA or 100 μM TSA substantially inhibited the voltage-gated Ca2+ current in these cells (Fig. 2, C and D).

Comparison of Inhibitory Potencies of TSA and SAHA on High Extracellular K+-Induced Ca2+ Entry with Their Vasodilatory Potencies.

We examined the effect of different doses of SAHA (3–300 μM) and TSA (1–100 μM) on Ca2+ influx induced by high extracellular K+. The results showed that SAHA and TSA caused dose-dependent inhibition on the high extracellular K+-induced Ca2+ influx with IC50 of 70 and 21 μM, respectively (Fig. 3, A and B, left panels).

Fig. 3.
Fig. 3.

Comparison of inhibitory potencies of SAHA and TSA on high extracellular K+-induced Ca2+ entry with their vasodilatory potencies. (A) Comparison of dose-response curves and IC50 values of SAHA on 60 mM KCl-induced cytosolic Ca2+ rise in smooth muscle cells (left panel) with vasodilatory action of SAHA in K+-contracted mouse aortas (right panel). (B) Comparison of dose-response curves and IC50 values of TSA on 60 mM KCl-induced cytosolic Ca2+ rise in smooth muscle cells (left panel) with vasodilatory action of TSA in K+-contracted mouse aortas (right panel). The data are expressed as the mean ± S.E.M. (n = 3–6).

Dose-dependent vasodilatory curves of SAHA and TSA on high K+-precontracted mouse aortas were also plotted (Fig. 3, A and B, right panels). The vasodilatory potencies of SAHA and TSA were similar to their inhibitory potencies on Ca2+ influx with comparable IC50 values (Fig. 3).

HDAC Inhibitors Reversed the Vascular Contraction Elicited by Phenylephrine in Mouse Aortas.

SAHA at 3–300 μM or TSA at 1–100 μM also induced dose-dependent relaxation of mouse aortas precontracted with an α-adrenergic receptor agonist phenylephrine at 1 μM (Fig. 4, A and B). Compared with the high K+-contracted arteries, the relaxant effect of SAHA and TSA was greater in phenylephrine-contracted arteries. Three hundred micromolar SAHA and 100 μM TSA almost completely reversed the phenylephrine-induced contraction (Fig. 4, A and B). Several other HDAC inhibitors, including PDX-101, LBH-589, and FK-228, also showed similar dose-dependent relaxation in phenylephrine-contracted arteries (Supplemental Fig. 1). In addition, the relaxant responses to SAHA and TSA were not significantly altered by endothelium denudation, suggesting that SAHA and TSA acted on vascular smooth muscle cells to exert their relaxant effect (Fig. 4, A and B).

Fig. 4.
Fig. 4.

HDAC inhibitors reversed the vascular contraction elicited by phenylephrine (Phe) in mouse aortas. Shown were vascular relaxant effect of SAHA (A) and TSA (B) on phenylephrine (1 μM)-precontracted aortas with endothelium (+Endo) or without endothelium (−Endo). Representative traces (left) and data summary (right) of relaxing effect are shown. The data are expressed as the mean ± S.E.M. (n = 5).

Relative contribution of L-type Ca2+ channels versus other components in the phenylephrine-precontracted arteries was estimated. Application of 10 μM verapamil caused 42–55% relaxation in these arteries (Fig. 5, A and B). Subsequent addition of 300 μM SAHA or 100 μM TSA resulted in further relaxation to nearly 100% (Fig. 5, A and B). These data suggest that L-type Ca2+ channels accounted for 42–55% of the contraction and the remaining 45–58% was independent of L-type Ca2+channels.

Fig. 5.
Fig. 5.

Relative contribution of L-type Ca2+ channels vs. other components in the phenylephrine-precontracted arteries. The aortas were precontracted with 1 μM phenylephrine (Phe), followed by 10 μM verapamil. After the relaxant response to verapamil became stable, 200 μM SAHA (A) or 100 μM TSA (B) was added to induce further relaxation. Representative traces (left) and data summary (right) of relaxing effect are shown. The data are expressed as the mean ± S.E.M. (n = 5). ***P < 0.001 vs. vehicle (DMSO) control.

SOCE Did Not Participate in the HADC Inhibitor-Induced Vascular Relaxation, but HADC Inhibitors Reduced the Phenylephrine-Induced Cytosolic Ca2+ Rise.

The mechanisms of vascular contraction in response to vascular agonists are complicated, involving Ca2+ release from intracellular Ca2+ stores, Ca2+ entry through store-operated Ca2+ influx and/or receptor-operated Ca2+ channels and/or L-type Ca2+ channels, as well as a sensitized contractile response to Ca2+ (Wang et al., 2008). We next explored whether HADC inhibitors could act on other mechanism(s) independent of L-type Ca2+ channels to induce vascular relaxation.

Calcium release-activated calcium channel protein 1 (Orai1) is the pore-forming subunit of SOCE in vascular smooth muscle cells (Lewis, 2011). As expected, the selective Orai1 inhibitor RO2959 at 200 nM (Chen et al., 2013) greatly inhibited the SOCE in the acutely isolated mouse smooth muscle cells (Fig. 6A). However, RO2959 up to 5 μM failed to induce relaxation in these arteries (Fig. 6B).

Fig. 6.
Fig. 6.

SOCE did not contribute to the HADC inhibitor-induced vascular relaxation. (A) Effect of RO2959 on SOCE in the freshly isolated mouse smooth muscle cells. The cells were treated with 4 μM thapsigargin (TG) in a Ca2+-free bath, which induced the first Ca2+ rise, followed by adding back 2 mM Ca2+ to the bath, which induced the second Ca2+ rise. The second Ca2+ rise was taken as SOCE. RO2959 at 200 nM was applied 5 minutes before TG. The representative traces (left) and data summary (right) of Ca2+ responses are shown. The summary data are expressed as the mean ± S.E.M. (n = 5 experiments, >25 cells per experiment). ***P < 0.001. (B) A representative trace showing lack of acute relaxant effect of RO2959 on 1 μM phenylephrine (Phe)-precontracted aortas. A representative time-control experiment (without RO2959) is displayed on top. n = 4 experiments.

In the absence of extracellular Ca2+, application of 1 μM phenylephrine failed to elicit a cytosolic Ca2+ rise (Fig. 7A), suggesting no significant stimulating effect of phenylephrine on intracellular Ca2+ release. Subsequent Ca2+ addback (2 mM) to the bath solution resulted in a cytosolic Ca2+ rise, which was more marked in phenylephrine-treated cells than in DMSO-treated vehicle control (Fig. 7A, left panel), suggesting that phenylephrine elicited extracellular Ca2+ entry. As there was no obvious intracellular Ca2+ release in response to phenylephrine (Fig. 7A), this Ca2+ entry may reflect receptor-operated Ca2+ entry rather than SOCE. Importantly, in the presence of 100 μM TSA or 300 μM SAHA, the phenylephrine-elicited extracellular Ca2+ entry was abolished (Fig. 7A).

Fig. 7.
Fig. 7.

Action of HDAC inhibitors on phenylephrine-induced cytosolic Ca2+ rise and comparison with their vasodilatory effect. (A) The freshly isolated mouse vascular smooth muscle cells were treated with 1 μM phenylephrine (Phe) in a Ca2+-free bath, followed by adding back 2 mM Ca2+ to the bath, which induced a Ca2+entry. SAHA/TSA at 100 μM was applied 2 minutes before Phe. The representative traces (left) and data summary (right) of Ca2+ responses are shown. (B) Comparison of dose-response curves and IC50 values of SAHA on Phe-induced cytosolic Ca2+ rise in smooth muscle cells (left panel) with vasodilatory action of SAHA in Phe-contracted mouse aortas (right panel). (C) Comparison of dose-response curves and IC50 values of TSA on Phe-induced cytosolic Ca2+ rise in smooth muscle cells (left panel) with vasodilatory action of TSA in Phe-contracted mouse aortas (right panel). The summary data are expressed as the mean ± S.E.M. [n = 5 experiments, >25 cells per experiment in (A); n = 3–5 in (B and C)]. **P < 0.01; ***P < 0.001.

Dose-dependent inhibition of SAHA (3–300 μM) and TSA (1–100 μM) on phenylephrine-induced Ca2+ influx was determined (left panels in Fig. 7, B and C). Dose-dependent vasodilatory curves of SAHA and TSA on phenylephrine-precontracted mouse aortas were also plotted (right panels in Fig. 7, B and C). The inhibitory potencies of SAHA and TSA on Ca2+ influx were similar to their vasodilatory potencies with comparable IC50 values (Fig. 7, B and C).

Lack of Nitric Oxide Involvement in TSA and SAHA-Induced Vascular Relaxation.

Methylene blue is a guanylate cyclase inhibitor commonly used to abolish nitric oxide–mediated vascular dilation (Gruetter et al., 1981). Preincubation with 10 μM methylene blue for 15 minutes had no effect on SAHA-induced vascular relaxation in mouse aortas contracted with high extracellular K+ (Fig. 8A) or phenylephrine (Fig. 8B), suggesting lack of nitric oxide involvement in SAHA-induced vascular relaxation.

Fig. 8.
Fig. 8.

Nitric oxide did not contribute to HADC inhibitor-induced vascular relaxation. (A) The aortas were precontracted with 60 mM KCl in the absence (upper left panel) or presence (lower left panel) of 10 μM methylene blue (MB). After the contractile response became stable (∼10 minutes), SAHA was added to induce relaxation. (B) The aortas were precontracted with 1 μM phenylephrine in the absence (upper left panel) or presence (lower left panel) of 10 μM methylene blue (MB). After the contractile response became stable (∼10 minutes), SAHA was added to induce relaxation. Shown are representative traces (left) and data summary (right). The data are expressed as the mean ± S.E.M. (n = 5).

Discussion

The main findings of the present study were as follows: 1) Two commonly used HDAC inhibitors, SAHA and TSA, partially reversed the high extracellular K+-induced contraction in mouse aortas. The effect of SAHA and TSA was acute, dose-dependent, but endothelium-independent. 2) SAHA and TSA also caused dose-dependent relaxation in phenylephrine-precontracted mouse aortas. Three hundred micromolar SAHA and 100 μM TSA completely reversed the phenylephrine-induced contraction. Again, the effect of SAHA and TSA was acute and endothelium-independent. 3) In the freshly isolated mouse smooth muscle cells, SAHA and TSA markedly reduced the high K+-induced cytosolic Ca2+ rise. Whole-cell patch-clamp studies demonstrated that SAHA and TSA acutely inhibited the voltage-gated L-type Ca2+ channel current in these cells. 4) SAHA and TSA also markedly reduced the phenylephrine-induced Ca2+ entry with inhibitory potency like their vasodilatory effect. 5) A potent Orai1 inhibitor, RO2959, failed to induce vascular relaxation in phenylephrine-induced contraction of mouse aortas. Taken together, the present study demonstrated that hydroxamate-based pan-HDAC inhibitors SAHA and TSA at intermediate micromolar concentrations could acutely inhibit L-type Ca2+ channels and consequently reduce the high extracellular K+-induced contraction of aortas. SAHA and TSA also relaxed the phenylephrine-precontracted aortas, a minor part of which could be attributed to the inhibitory effect of these compounds on L-type Ca2+ channels.

It is well documented that exposure to high extracellular K+ at 60–100 mM causes vascular contraction (Eto et al., 2001). The underlying mechanism involves high K+-induced membrane depolarization of vascular smooth muscle cells, which triggers an opening of voltage-gated Ca2+ channels, especially L-type Ca2+ channels. Subsequent Ca2+ influx through L-type Ca2+ channels elevates cytosolic Ca2+ level in vascular smooth muscle cells, resulting in vascular contraction (Eto et al., 2001). In the present study, high extracellular K+-induced aortic contraction was completely reversed by 10 μM verapamil, confirming the involvement of L-type Ca2+ channels. Importantly, SAHA and TSA partially but acutely reversed the high extracellular K+-induced contraction of mouse aortas, suggesting a possible inhibitory action of these agents on voltage-gated Ca2+ channels. Two sets of experiments, cytosolic Ca2+ measurement and whole-cell patch-clamp, were performed to verify the action of SAHA and TSA on L-type Ca2+ channels. In cytosolic Ca2+ measurement, high extracellular K+-evoked cytosolic Ca2+ rise was reduced by SAHA and TSA. Whole-cell patch-clamp experiments provide the direct evidence for inhibitory action of SAHA and TSA on L-type Ca2+ channel current in vascular smooth muscle cells. Interestingly, inhibitory potencies (IC50) of SAHA and TSA on high extracellular K+-induced Ca2+ rise were similar to the vasodilatory potencies of these compounds in high K+-contracted arteries. Taken together, these results provided unequivocal evidence for inhibitory action of HDAC inhibitors on the activity of L-type Ca2+ channels in vascular smooth muscle cells.

Our finding about the modulatory effect of HDAC inhibitors on voltage-gated Ca2+ channels has important physiologic and pathologic implication. SAHA and several other HDAC inhibitors are in clinical use for cancer treatment (Zwergel et al., 2016). In animal models, HDAC inhibitors also show beneficial cardiovascular effect by alleviating cardiac hypertrophy, hypertension, and myocardial infraction (Eom and Kook, 2014). In the present study, we show for the first time that HDAC inhibitors can acutely inhibit L-type Ca2+ channels and subsequently reduce the vascular tone. It is well documented that L-type Ca2+ channels are closely associated with vascular tone and that an aggravated activity of L-type Ca2+ channels is a causative factor for pathologic progression of hypertrophy, hypertension, and myocardial infraction (Nayler, 1993). Therefore, our present results about the effect of HDAC inhibitors on L-type Ca2+ channels may provide a mechanistic explanation for cardio-protective effect of HDAC inhibitors against hypertrophy, hypertension, and myocardial infraction.

The mechanisms of vascular contraction in response to vascular agonists such as phenylephrine are complicated. These may include Ca2+ release from intracellular Ca2+ stores, Ca2+ entry through store-operated Ca2+ influx and/or receptor-operated Ca2+ channels and/or L-type Ca2+ channels, as well as a sensitized contractile response to Ca2+ (Nishimura et al., 1989; Sun et al., 2000). In our study, SAHA and TSA could completely reverse the phenylephrine-induced contraction in mouse aorta. As expected, part of the phenylephrine-induced contraction, which amounts to 42–55%, was mediated by L-type Ca2+ channels (Nishimura et al., 1989), as indicated by its sensitivity to verapamil inhibition. However, in the presence of verapamil, SAHA and TSA could cause additional relaxation, which presumably resulted from other mechanisms independent of L-type Ca2+ channels. In our studies, phenylephrine failed to elicit significant intracellular Ca2+ release (Fig. 7A). Furthermore, inhibition of SOCE by an Orai1 inhibitor RO2959 failed to reverse the phenylephrine-induced contraction (Fig. 6B). These data suggest that the vascular relaxant effect of SAHA and TSA was independent of intracellular Ca2+ release or SOCE. Nevertheless, SAHA and TSA could inhibit phenylephrine-elicited Ca2+ entry. Furthermore, inhibitory potencies (IC50) of SAHA and TSA on phenylephrine-induced Ca2+ entry were similar to the vasodilatory potencies of these compounds in phenylephrine-contracted arteries, suggesting an important role of cytosolic Ca2+ in these events. Together, these data suggest a possible action of HDAC inhibitors on some Ca2+-signaling mechanisms independent of SOCE and intracellular Ca2+ release. These may include receptor-operated Ca2+ entry, Ca2+ sensitivity of contractile mechanism, or other alternative mechanisms.

One concern is that under oxidative stress, hydroxamate compounds, including SAHA and TSA, are able to produce nitric oxide (Samuni et al., 2009), which could account for the vasodilatory action of these compounds. However, all our experiments, including vascular tone studies and Ca2+ entry and patch-clamp experiments, were performed under normal physiologic saline without oxidative stress. Indeed, pretreatment of arteries with methylene blue, a guanylate cyclase inhibitor, had no effect on SAHA-induced vascular dilation, suggesting that the vasodilatory response to hydroxamate compounds in our experiments was not the result of hydroxamate-generated nitric oxide.

SAHA and TSA are pan-HDAC inhibitors that inhibit class I and class II HDAC proteins. However, their inhibitory potency to different classes of HDAC proteins varies greatly (Dokmanovic et al., 2007; Ko et al., 2013; Lobera et al., 2013). The reported IC50 values of SAHA and TSA for class I and class IIb HDAC proteins range from nanomolar to sub-micromolar, whereas the IC50 values for class IIa HDAC proteins range from low to intermediate micromolar (Dokmanovic et al., 2007; Ko et al., 2013; Lobera et al., 2013). In our studies, SAHA and TSA relaxed the extracellular high K+- and phenylephrine-precontracted aortas at intermediate micromolar concentration, which could inhibit class IIa HDAC proteins. However, considering the issue of drug permeation in cultured cells and vascular tissue, it is still uncertain which specific class of HDAC proteins might be involved.

In conclusion, we have demonstrated that HDAC inhibitors can cause acute inhibition of the activity of L-type Ca2+ channels, resulting in partial reversal of high extracellular K+-induced vascular contraction. HDAC inhibitors also acutely and potently reverse the phenylephrine-induced vascular contraction via L-type Ca2+ channel-dependent and -independent mechanisms.

Acknowledgments

The authors thank Dr. Jiangyun Luo, Zhiheng Liu, and Cheng Chen for comments that greatly improved the manuscript.

Authorship Contributions

Participated in research design: Zheng, Zhong, Huang, Tsang, Yao.

Conducted experiments: Zhong, Zheng, Qi, Shen, Zhao, Wu.

Performed data analysis: Zhong, Zheng, Qi.

Wrote or contributed to the writing of the manuscript: Yao, Zhong, Zheng, Qi, Huang, Tsang.

Footnotes

    • Received May 7, 2017.
    • Accepted August 21, 2017.

  • ↵1 C.Z. and M.Z. contributed equally to this work.

  • This work was supported by grants from Hong Kong Research Grant Committee (No. AoE/M-05/12, 14118516), RGC-NSFC Joint Grant (No. N_CUHK439/13) and National Natural Science Foundation of China (No. 81070066, 31470912).

  • https://doi.org/10.1124/jpet.117.242685.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

BAPTA
1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
DMSO
dimethyl sulfoxide
HDAC
histone deacetylase
Orai1
calcium release-activated calcium channel protein 1
p66shc
Src homology 2 domain-containing transforming protein C1, MW 66 isoform
RO2959
2,6-difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide hydrochloride
SAHA
suberoylanilide hydroxamic acid
SOCE
store-operated Ca2+ entry
TSA
trichostatin A
U46619
(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid

This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

References

  1. ↵
    1. Chen G,
    2. Panicker S,
    3. Lau KY,
    4. Apparsundaram S,
    5. Patel VA,
    6. Chen SL,
    7. Soto R,
    8. Jung JKC,
    9. Ravindran P,
    10. Okuhara D, et al.
    (2013) Characterization of a novel CRAC inhibitor that potently blocks human T cell activation and effector functions. Mol Immunol 54:355–367.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Choudhary C,
    2. Kumar C,
    3. Gnad F,
    4. Nielsen ML,
    5. Rehman M,
    6. Walther TC,
    7. Olsen JV, and
    8. Mann M
    (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Colussi C,
    2. Scopece A,
    3. Vitale S,
    4. Spallotta F,
    5. Mattiussi S,
    6. Rosati J,
    7. Illi B,
    8. Mai A,
    9. Castellano S,
    10. Sbardella G, et al.
    (2012) P300/CBP associated factor regulates nitroglycerin-dependent arterial relaxation by N(ε)-lysine acetylation of contractile proteins. Arterioscler Thromb Vasc Biol 32:2435–2443.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Dokmanovic M,
    2. Clarke C, and
    3. Marks PA
    (2007) Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res 5:981–989.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Eom GH and
    2. Kook H
    (2014) Posttranslational modifications of histone deacetylases: implications for cardiovascular diseases. Pharmacol Ther 143:168–180.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Eto K,
    2. Ohya Y,
    3. Nakamura Y,
    4. Abe I, and
    5. Fujishima M
    (2001) Comparative actions of insulin sensitizers on ion channels in vascular smooth muscle. Eur J Pharmacol 423:1–7.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Griffith DM,
    2. Szocs B,
    3. Keogh T,
    4. Suponitsky KY,
    5. Farkas E,
    6. Buglyó P, and
    7. Marmion CJ
    (2011) Suberoylanilide hydroxamic acid, a potent histone deacetylase inhibitor; its X-ray crystal structure and solid state and solution studies of its Zn(II), Ni(II), Cu(II) and Fe(III) complexes. J Inorg Biochem 105:763–769.
    OpenUrlPubMed
  8. ↵
    1. Gruetter CA,
    2. Gruetter DY,
    3. Lyon JE,
    4. Kadowitz PJ, and
    5. Ignarro LJ
    (1981) Relationship between cyclic guanosine 3′:5′-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin. J Pharmacol Exp Ther 219:181–186.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Hebbel RP,
    2. Vercellotti GM,
    3. Pace BS,
    4. Solovey AN,
    5. Kollander R,
    6. Abanonu CF,
    7. Nguyen J,
    8. Vineyard JV,
    9. Belcher JD,
    10. Abdulla F, et al.
    (2010) The HDAC inhibitors trichostatin A and suberoylanilide hydroxamic acid exhibit multiple modalities of benefit for the vascular pathobiology of sickle transgenic mice. Blood 115:2483–2490.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Kang G,
    2. Lee YR,
    3. Joo HK,
    4. Park MS,
    5. Kim CS,
    6. Choi S, and
    7. Jeon BH
    (2015) Trichostatin a modulates angiotensin II-induced vasoconstriction and blood pressure via inhibition of p66shc activation. Korean J Physiol Pharmacol 19:467–472.
    OpenUrl
  11. ↵
    1. Ko KS,
    2. Steffey ME,
    3. Brandvold KR, and
    4. Soellner MB
    (2013) Development of a chimeric c-Src kinase and HDAC inhibitor. ACS Med Chem Lett 4:779–783.
    OpenUrl
  12. ↵
    Lewis RS (2011) Store-operated calcium channels: new perspectives on mechanism and function. Cold Spring Harb Perspect Biol 3(12) DOI: 10.1101/cshperspect.a003970.
  13. ↵
    1. Mathews KS,
    2. Corso P,
    3. Bacon S, and
    4. Jenq GY
    (2014) Using the red/yellow/green discharge tool to improve the timeliness of hospital discharges. Jt Comm J Qual Patient Saf 40:243–252.
    OpenUrl
  14. ↵
    1. Nayler WG
    (1993) Pharmacological aspects of calcium antagonism. Short term and long term benefits. Drugs 46 (Suppl 2):40–47.
    OpenUrl
  15. ↵
    1. Nishimura J,
    2. Khalil RA, and
    3. van Breemen C
    (1989) Agonist-induced vascular tone. Hypertension 13 (6 Pt 2):835–844.
    OpenUrl
  16. ↵
    1. Ochi R,
    2. Dhagia V,
    3. Lakhkar A,
    4. Patel D,
    5. Wolin MS, and
    6. Gupte SA
    (2016) Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells. Am J Physiol Heart Circ Physiol 310:H1118–H1128.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Pandey D,
    2. Sikka G,
    3. Bergman Y,
    4. Kim JH,
    5. Ryoo S,
    6. Romer L, and
    7. Berkowitz D
    (2014) Transcriptional regulation of endothelial arginase 2 by histone deacetylase 2. Arterioscler Thromb Vasc Biol 34:1556–1566.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Qi Y,
    2. Qi Z,
    3. Li Z,
    4. Wong C-K,
    5. So C,
    6. Lo I-C,
    7. Huang Y,
    8. Yao X, and
    9. Tsang S-Y
    (2015) Role of TRPV1 in the differentiation of mouse embryonic stem cells into cardiomyocytes. PLoS One 10:e0133211.
    OpenUrl
  19. ↵
    1. Richardson PG,
    2. Moreau P,
    3. Laubach JP,
    4. Maglio ME,
    5. Lonial S, and
    6. San-Miguel J
    (2017) Deacetylase inhibitors as a novel modality in the treatment of multiple myeloma. Pharmacol Res 117:185–191.
    OpenUrl
  20. ↵
    1. Samuni Y,
    2. Flores-Santana W,
    3. Krishna MC,
    4. Mitchell JB, and
    5. Wink DA
    (2009) The inhibitors of histone deacetylase suberoylanilide hydroxamate and trichostatin A release nitric oxide upon oxidation. Free Radic Biol Med 47:419–423.
    OpenUrlPubMed
  21. ↵
    1. Sun L,
    2. Fan JS,
    3. Clark JW, and
    4. Palade PT
    (2000) A model of the L-type Ca2+ channel in rat ventricular myocytes: ion selectivity and inactivation mechanisms. J Physiol 529:139–158.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Tian XY,
    2. Wong WT,
    3. Wang N,
    4. Lu Y,
    5. Cheang WS,
    6. Liu J,
    7. Liu L,
    8. Liu Y,
    9. Lee SST,
    10. Chen ZY, et al.
    (2012) PPARδ activation protects endothelial function in diabetic mice. Diabetes 61:3285–3293.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Usui T,
    2. Okada M,
    3. Mizuno W,
    4. Oda M,
    5. Ide N,
    6. Morita T,
    7. Hara Y, and
    8. Yamawaki H
    (2012) HDAC4 mediates development of hypertension via vascular inflammation in spontaneous hypertensive rats. Am J Physiol Heart Circ Physiol 302:H1894–H1904.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Wang Y,
    2. Deng X,
    3. Hewavitharana T,
    4. Soboloff J, and
    5. Gill DL
    (2008) Stim, ORAI and TRPC channels in the control of calcium entry signals in smooth muscle. Clin Exp Pharmacol Physiol 35:1127–1133.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Zhang J,
    2. Berra-Romani R,
    3. Sinnegger-Brauns MJ,
    4. Striessnig J,
    5. Blaustein MP, and
    6. Matteson DR
    (2007) Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+. Am J Physiol Heart Circ Physiol 292:H415–H425.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Zwergel C,
    2. Stazi G,
    3. Valente S, and
    4. Mai A
    (2016) Histone deacetylase inhibitors : updated studies in various epigenetic - related diseases. J Clin Epigenetics 2:1–15.
    OpenUrl
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HDACi Relax Mouse Aorta through L-Type Ca2+ Channel

Changbo Zheng, Mingkui Zhong, Zenghua Qi, Fan Shen, Qiannan Zhao, Lulu Wu, Yu Huang, Suk-Ying Tsang and Xiaoqiang Yao
Journal of Pharmacology and Experimental Therapeutics November 1, 2017, 363 (2) 211-220; DOI: https://doi.org/10.1124/jpet.117.242685
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